CA1322078C - Enzyme inhibitors - Google Patents

Abstract

ABSTRACT
Peptide analogues of formula (I) wherein X and W are terminal groups optionally including further amino acyl groups; Y where present is glycine phenylalanine or other lipophilic amino acid residue; Z is glycine N-methyl alanine valine proline or a ring homologue of proline; A is a 'hydroxy' 'reduced' or preferably 'keto' dipeptide analogue residue of which the first residue is either arginine or has the side chain termi-nal amidino group of arginine and the second is a glycine alanine or related residue with a hydrocarbon side chain optionally hydroxy terminated; Arg or Pro and Arg where present are option-ally substituted; and B where present is valine proline or a group -NH-(CH2)n-CO- (n = 0 - 5), or with W represents an aminoalcohol;
and wherein optionally the terminal groups are linked to give a cyclic structure are useful inhibitors of thrombogenesis.

Description

1322~78 ENZYME INHIBITION

The invention relates to peptide analogues and to the inhibition of thrombogenesis.
Thromb~enesis S Blood clotting depends on a complex series of actions leading finally to cleavage of fibrin from circulating fibrinogen by the protease thrombin, and its subsequent cross linking to form a stable structure. Thrombin production falls in two parts, an 'intrinsic' system based on circulating blood components and an 'extrinsic' system requiring tissue components. In each, there is a cascade of reactions, a series of inactive 'factors' each being converted by proteolytic reaction into the corresponding 'a' factor ~hat is itself a proteolytic enzyme effecting the next step.
In the intrinsic system the actions start ~ith circulating Hageman factor (factor XII) undergoing contact activation, becoming bound to damaged surfaces or plate~t aggregations. A peptide is cleaved from it by the circulating protease kallikrein, forming factor XIIa. Factor XIIa in the presence of circuLating high molecular weight kininogen (HMW-K~ then a~ cleaves circulating plasma thromboplastin antecedent (factor XI) to give plasma thromboplastin (factor XIa) itself and b) cleaves circulating pre-kallikrein (Pre-K, Fletcher factor) giving more kallikrein and thus a self-accelerating action. Plasma thromboplastin, in the presence of calcium ions, cleaves circulating Christmas factor (factor IX~ to give factor IXa. Factor IXa, with circ~llatiDg antihaemophilic globulin (AHG, factor VIII) and in ~he presence of calcium ions and phospholipid micelles from platelets, forms a lipoprotein complex with circulating Stuart factor (factor X) and cleaves it to form factor Xa. Finally factor Xa, with circulating labile factor (factor V) and in the presence of calcium ions and phospholipid micelles, forms a lipoprotein complex with prothrombin (factor II) and cleaves it to form thrombin (factor IIa) itself.
In the extrinsic system, a serum glyco-protein proconvertin (factor VII) is cleaved by several proteases from the intrinsic system (factors XIa and XIla and kallikrein~to form factor VIIa. Factor VIIa, with a tissue lipoprotein called tissue thromboplastin (factor III), and in the presence of calcium ions, forms a complex with further circulating factor X and cleaves it to form a second source of factor Xa, enhancing thrombin production.

Finally, thrombin from both sources converts circulating fibrinogen to the soluble form of fibrin, which spontaneously polymerizes into filaments and is then cross linked under the action of an enzyme (factor XIIIa) formed, by a second action of throm~ln, from circulating fibrin stabilising factor (factor XIII).
In t~bular form the accelerating cascade of actions is:

1322~78 o C V
C

C

K~ K
0 ,~ C CO C 0 1~

qo~ I 0 K K ~ ~ 8 ~ C ~
~ ~ ~ ~ ~ c 1 ~ c ~ ~ c 01 ~ ~ C ~ ~ E C-. ~ V

C ~

il -- O
~_ Vo ~; 0 C 0 V

VK¦ V ~ 3 K ~¦
0~ 0 I ~C C~o g Cl zo ~læ~

1~22~78 Thrombin Besides its central action in fibrin clot formation set out ~bove, thrombin i8 a key factor in the platelet aggregation state of thrombogenesis as discussed for example in H.J. Weiss "Platelets:
Pathophysiology and Antiplatelet Drug Therapy" Ch. 1 (Liss, New York, 1982) or D. Ogsten and B. Bennett (Eds.) "Haemostasis :
Biochemistry, Physiology and Pathology" Ch. 13 (Wiley, 1977).
The details are not fully understood, but thrombin is the strongest inducer ~f the 'platelet reaction' (shape change, aggregation, dense granule and ~-granule secretion) responsible for primary haemostasis. The action of thrombin is preceded by its interaction with ~ speclf~c b~ndi~g protein ~n the platelet membrane, and the inhibitors of the present invention also appear to interfere with this binding.
Specific inhibitors of thrombin are thus to be expected to act as highly effective antithrombotic agents and/or anticoagulants, giving alternatives to existing therapeutic and prophylactic agents. Heparin is for example videly used against thrombosis, but it carries a high risk of haemorrhage, is ineffective in many cnn~tions, and gives a variable respouse from patient to patient and from t~me to time in a given pHtient ~o that careful monitoring is required. Oral anticoagulants also, used particularly for proph~laxis and control of venous thrombosis, take time to develop their effect and are interfered with by many other drugs. The inhibitors of the present invention act at omce, ~nd because of their spec;~iclty and chemical nature may be expected to be without side effects or drug interactions.
Further, by acting only on the ths3~bin erigger to platelet aggregation, they leave the colLagen trigger available to preserve haemostasis. Administration is simple, for example intranasal.

1322~78 Substrate Structure It is known that most of the affinity sites for thrombin are present in the N-terminal (8-20) fra~ment of fibrinogen, and tha~ partial sequences of this region bind tightly to the enzyme and are rapidly hydrolysed by it. Specifically,thrombin acts on the A ~-chain of fibrinogen ~ ~'J
Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val between Argl6and Glyl7, to remove the N-terminal hexadecapeptide fragment (fibrinopeptide A). Subsequently, cleavage between residues Argl9 and Val20, again by thrombin, takes place at a much slower rate, releasing a further tripeptide fragment. The enzyme also removes the N-terminal tetradecapeptide fragment (fibrinopeptide B) of the fibrinogen B~ chain by slow hydrolysis between Argl4 and Gly . This release of the fibrinope~tides from fibrinogen is followed by polymerisation of the resulting fibrin monomer to form "soluble"fibrin. The latter is then converted into stable, "insoluble" fibrin gel by Factor XIIIa, which cross-links two ~ or ~r-chains of neighbouring fibrin lecules by transamidation.
THE INVENTION
_ The present invention is based on partial sequences of human fibrinoge~ which possess high binding affinity for thrombin. These substrate fragments are modified by replacement of the scissile peptide bond -CO-NH-~ 322G78 ~6-(i.e. the bond normally cleaved by thrombin) with a non-hydrolysable isosteric linkage (e.g. a keto -CO-CH2-, hydro~y -CH(OH)-CH2 or reduced -CH2-Nnl- linkage).

Optionally, other positions of the substrate sequence are also modified to provide increased binding affinity, e.g. by introducing DPhe at position 14 and/or Pro at position 15 of fibrinoge~ as previously proposed in itself by M. Pozsgay et al. Eur. J. Biochem 115 941 (1981), or increased metabolic stability, e.g. by 1 isosteric substitution of additional peptide linkages or by protection of the N-terminus and/or C-terminus with suitable protecting groups.
General refereDce to amino acids and amino acyl residues and side-chains in both the description and claims, is to be taken as reference to fiuch whether naturally occurring in proteins or not, and to both D-and L- forms; and amino acid is to be taken as including imino a~id. All asymmetric centres may be of either R
or S configurati~n unless stated otherwise.
The coopoonds of the present invention, showing desirable anti-~hIombotic or anti-coagulant action, are of the general formula I below (wherein numbering of residues correspondsto that in human fibrinogen, but without limitation of the invention):

~322~78 X - Y - Z A - Pro- Arg B W (I) 14 15 16,17 18 19 20 wherein X and W are terminal groups optionally including further amino acyl groups; Y where present is glycine phenylalanine or other lipophilic amino acid residue; Z
is glycine N-methyl alanine valine proline or a ring homologue of proline; A is a 'hydroxy' 'reduced' or preferably 'keto' dipeptide analogue residue of which the first residue is either arginine or has the side chain terminal amidino group of arginine and the second is a glycine alanine or related residue with a hydrocarbon side chain optionally hydroxy terminated; Arg or Pro and Arg where present are optionally substituted; ~nd B where present is valine proline or a group -NH-(CH2)n-CO-(n ~ O - 5), or with W represents an aminoalcohol;
and wherein optionally the terminal groups ~re linked to give a cyclic structure.
In more detail, in formula I desirably:
X = H or a protective group including lower alkyl (Cl-C5), or lower aliphatic acyl ~Cl-C5),or aromatic acyl, e.g. Ar~(CH2)n-CO- or Ar-O-(CH2)n-CO- where n - O - 2 and Ar is phenyl or other (including mono- or bicyclic) aromatic group which may be substituted especially mono-substituted with one of the following groups, preferably (when p~enyl) in the 2- or 4-position :
F, Cl, Br, I, -CF3, -OH, -OR or -R (R = Cl-C6 alkyl) 1322~78 or R-0-C0- where R = t butyl, Ar-alkyl (e.g.benzyl), 2,2,2-trichloroethyl or R -S02- where R ~ Ar (e.g. Ph, c~-naphthyl, ~-naphthyl) or other lipophilic group or one or more amino acyl residues either as such or in protected form bearing a group X above Y = absent, or glycine, or D- or L-phenylalanine or other lipophilic amino acid re~idue (e.g.
Phg, Cha, ~-Nal, ~-Nal, p-iodophenylalanyl) Z = L- or D-proline or a ring ho~ologue (e.g.
azetidine- 2-carboxylic acid, piperidine-2-carbo ylic acid) or L- or D-valine or N-methyl-alanine or glycine or Y and ~ are as:
~7z ~here the peptide bond -CO-NH- between Y and Z
~ has been reduced (and protected) to give -CH2-N¦X)-, X being a protective group as defined above g A - (1) Rl ~2 -NH-CH-C-CH -CH-CO- Iketo' isostere (II) 2 *

~2) Rl R2 ,~ I
-NH-CH-CH-CH2-CH-CO- "hydro~y'isostere ~III) OH

(3) R R3R2 -NH-CH-CH~-N-CH-CO- 'reduced' isostere (IV) where:
(i) R = -(cH2)n-NH-c-NH2 with n = 2-4 NH
or ~ 2)m ~c~2 3 3 where m = 0-2 ~
or (c~ ~ 3 (ii) R2 = H, lower alkyl (Cl-C4) or l-hydro~y-ethyl, 132~78 (iii) R - any one of groups ~ as defined above (iv) configuration at the asymmetric centres *
is either R or S

Arg or Pro and Arg may be absent, Pro may be in S substituted form (e.g. hydroxy-proline) and Arg may be in substituted form (e.g. with zl, Tos or -N02) B = D or L-valine or D or L-proline or -NH-(CH2)n-CO-, where n = 0-5, or absent 0 W - -OH as such or in protected form including -oR4, where R4 ~ lower alkyl (Cl-C5) or -NH2 as such or in protected form including -NHR5 or -NR5 where R = lower alkyl (Cl-C5) or R2 s cycloalkyl -(CH2)n- with n = 3 - 5 where the ring is optionally substituted with carboxyl -COOH and/or alkyl (Cl-C5) group(s) B-W represents an aminoalcohol derivative of B as such or in protected form, or a residue in which one or more further amino acyl groups are present and where further in said compounds of formula I
(especially those equivalent to a hexapeptide) the two terminals are optionally linked by a peptide bond to form a cyclic structure.

1322~7~

Particularly in general formula I above the following may apply:

X ~ H, lower alkyl (Cl-C5) or other protecting group e.g. lower acyl (Cl-C5) or R-0-C0- where R
= t-butyl, benzyl, 2,292-trichloroethyl, or an amino acyl residue either as such or in a protected form Y = D- or L-phenylalanine, or glycine 0 Z = L- or D-proline, or L- or D-valine In A :- R = -(CH2)n-NH-~ - NH2 with n = 2, 3 or 4 NH
- R2 . H, methyl, isospropyl, sec-butyl, iso-butyl or l-hydroxy-ethyl - R3 = H, lower aliphatic acyl Cl-C5 or lower alkyl Cl-C5, or R-0-C0- as for X
- configuration at the asymmetric centres *
is either R or S

Pro and Arg, or Arg alone, may be absent, or in substitu~ed form e.g. OH for Pro, or Z (benzyloxycarbonyl) or N02 for Arg B ~ D- or L-valine, or D- or L-proline, or absent -12- 1322~78 W e -OH, or -oR4 where R4 - lower alkyl Cl-C5, or -NH2' or -NHR5 or NR52 where R5 = lower alkyl (Cl-C5) or R2 = lower cycloalkyl -(CH2)n- with n = 3, 4 or 5 Such peptide analogue may further be in the above form or may be modified by isosteric replacement of one or more remaining peptide bonds by keto -CO-CH2-, hydroxy -CH~OH)-CH2- or reduced -CH2-NH- linkages, and may be in the free form or in a protected form at one or more remaining functional groups e.g. amino, imino or amide (including peptide) nitrogen, carboxyl, hydroxyl, guanidino. In particular, the analogues may be present in the form of their physiologically acceptable acid addition salts or other derivatives convertible in the body to the active compound (as shown by their effect). Such physiologically acceptable derivatives are included within the definition of the compounds in the description and claims herein.
Protective or substituent groupings may be any of those known in the polypeptide art, amply disclosed in the literature and not requiring discussion here. Generally the selection of the groups is according to their function, some being primarily intended to protect against undesired reaction during synthetic procedures while the N- and C-terminal substituents are for example directed against the attack of exopeptidases on the final compounds or to ~13- 1 3 22 ~7 8 increase their solubility or lipophilicity and hence physiological acceptability. All these functions are generally within the term "protective group" or the like used in the description and claims herein~
The invention further extends to the use of the above described thrombin inhibitors in the prophylaxis or treatment of diseases associated with undesirable thrombo-genesis.

EXAMPLES

The following detailed Examples illustrate the invention, the text of the examples being followed by tabulated reaction schemes. Examples I to VI are given in detail, followed by more abbreviated Examples VII to XVI on the same lines.

Ex~mnle I

(DL)K
H - 163, Boc - DPhe - Pro - Arg - Gly - Pro - OH

The ~ynthe~iJ of H-163 wa3 carried out according to Scheme 1. Araoic numernl~ underllned (e.g. 1) refer to ~tructure3 in the~s Scheme~. Arabic numerAl~ ln parenthe~e6, e.8.(1) ro~er to reactlon ~tep~.
(1) Na-~ormyl-~G,N8-di(benzyloxycnr~:ionyl)-L-ar$inine 1 Yn~i obtnined from N~-formyl-L-arginine by the method o~ Smithwick and Shuman (J.Org.Chem. 1974.39.3~41) in 62% yield, or:by the 0 formylation of N-Arg(Z2)-0 X with formic-pivalic anbydride(60~).
(2) Oxnzolone 2. HCO-Ar4(Z2)-OH (1 mmol) was di~solved in dry THF-CH2C12 (1:5) and cyclised by treatment with DPECl.HCl ~alt ll.1 mmol) at O for 2 hrs. The oxazolone 2 wa~ isolated a~
n p~le yellow oil.
l5 (3)(4) nnd (5). The oxazolone 2 (1 mmol) in dry THF nt 0 WAS
acylated w~th the ~uccinic hemiestor chloride ~ (1.1 mmol) in tho pre~ence of Et3N (1.2 mmoI). The rcaction mixture was ~tirred ~t 2Z for 2 hrs, evaporated, the residue di~ol~ed in pyridine nnd treated with D~AP (20 mg) at 22 for 90 mins.
~cOH (2.5 ml) was added and the red ~olution wa~ left at 22 or 16 hrs~ After evaporatio2, and extraction of the crude produ~t with ethyl acetate, chromAtogrAphy on silicn with EtOAc-petrol yielded the pure trichloroethyl e~ter 6 a~ n colorle~s oil (27~ from 1) 5 (6) The trichloroethyl ester group wa~ removed rrom 6(~.2 ~mol~

~y trent~nt ~t O in THF (25 ml) ~ith Zn powder (2.4g) ~nd eold ~M N~H2P04 (6.5 ml) for 2~ hrs. The crude product was oxtracted ~ith EtOAc and crystalli&ed rrom EtOAc - petrol to give pure NCO-Arg(Z2)- Gly-OH, 7 ~82%~.

1322~78 (7) The prot~cted keto iso~ter- ? l- 189 ~mol) w~ converted into its Pfp e-tor by tre~t~ent with Pfp-OH (0.2 ol) ~nd DCCI (0.19 ~mol) in CH2C12 (2 ml) nt O for 1~ hr~. Thi~
Pfp e~ter ~ns coupled at O to H-Pro-OMe. HCl ~alt (1.1 mmol) ln DMF contnining Pr2 NEt (2.5 equivalents). ChromatogrAphy of the crude product in EtOAc on silica yielded pur~
HCO-Arg(Zz)-Gly-Pro-OMe o (80%) ~J a colorless oil.
(8)(9~ The formyl protecting group of 8 (0.13 mmol) was removed by treatment with 1 M HCl/MeOH (30 ml) at 22 for 16 hrs.
0 Evapor~tion yielded H-Arg(Z2)-Gly-Pro-OMe.HCl,9 which w~s dissolved in DMF nnd c~upled with Boc-DPhe-Pro-OPfp 11 ~0.144 ~mol) at O in the presence Or Pr2 NEt (0.27 ~nol).
The crude methyl ~ster 12 thus formed was purified by cbromatogrAphy on 8ilica ln EtOAc and obtained as a colorless oil (70%).
~10)(11) The methyl ester 12 (0.04 mmol) in MeOH (0;9 ml) was treated with 1 M ~OH (0.1 ml) for 1~ hr~.nt 22 to remove the e~ter group ~nd one of the Z protecting groups. The crude product w~s purified by hplc on Lichroprep RP18 in a gradicnt of MeOH and 5% AcOH to give the pure peptide acid 13 (26 mg).
The latter was d~s~olved in MeOH-ACOH-H20 (5:1:1) and hydrogenatea over 5% Pd~C to yield pure H-163. Tlc ~nd hplc ~how the pre~ence of two epimers. Tlc on ~ilica in CHC13-~eO~-ACOH (6:2:1) ~ 0.19 nnd 0.22. After hydroly~is ~t 110fl8 hrs. ~itb 6N HCl, amino ~cid nnalysis:
Pro, 2.0; Phe, 0.99.
Exam~le II
(DL)K
N - 173, H-DPhe - Pro - Arg - Gly - Pro - OH
(See Scheme I ) . * Trade ~ark 1322~78 (12) The N -~oc-protected peptide H-163 was treated ~-ith aqueous 2M HCl for 2 hr~ at 22. The re~ulting deprotected peptide H-173 wn~ obtalned by lyophili~ation. ~naly~
Pro, 2.05, Phe, 0.95.

ExamDle III

N - 170, Boc - DPhe Pro - Arg - Gly - Pro - NHEt (D) (see Schem~ II, V) (1~ The protected ~eto isostere 7 (0.189 ;mol) was converted into its Pfp ester ~nd coupled to H-Pro-NHEt (1.1 equi~alents~
in the came manner 8S described for the methyl ester in Ex~ple I ~tep (7), to give the ethylamide 15 in 95~ yield.
(2)(3) The formyl protecting group was removed from 15 by treatment ~ith lM HClJMeOH and the resulting N -deprotected peptide amide 16 wa~ ncylated with the Pfp e~ter 11 ns described in ~ectlons (8) and (9) of Exumple I, to give a mixture Or the epimers ~2~ and 17b (136 mg,75~).
~) The latter were ~eparated by chromatography on silica i~
~eOH-EtOAc, yielding 56 mg of the ~afiter moving epimer 17a (PF -35 on a silica tlc plate in MeOH-EtOAc 1:10) and 60 ~g of the Jlo~er one 17b (~ 0.30). Synthesis of 17 by an independent route which preserves optic~l integrity at the ~-cnrbon ~tom o~ L-arginine (see Scheme V) gave the sloY
moving epimer ~, thu~ enabling a3~ignment of confi~ur~tion in tho eplmers 17a (fa~t moving, contain~ D-arginin4) ~nd ~ lo~-moving, contains L-~rginine). Hydro~enol~i~ o-17a ov~r 5% Pd~C yielded the ~oc-protected pentapepti~e ethylcmide H-1?0. Tlc on silic~ in CHCl3-~leOH-AcOH (6:2:1~
0.39. Amino scid enalysi9 fter hydrolysis ~t 110~lB hrs.
~ith 6N HCl: Pro 2.04; Phe o.96.

~322~7~

Ex~ple IV

H - 179, ~ - DPhe - Pro - ~rg - Gly - Pro - ~NEt (L) (~ee Scheme II) (4) Hydrogenoly~iJ of the ~low mo~ing epimer 17b, o~tained in Jtep (3) of the ~yntho~i of H-170, produced the Boc-pontnpeptido Dmide H-171. The latter wa~ treated ~ith 2N HCl at 22 for 2 hrs and lyophilised to yield ~-179.
Tlc on ~lica in C~C13-MeOH-AcOH (6:2:1) R~ 0.15.
~ino acid ~nly~is: Pro, 2.00; Phe 1.00 0 Exnmnle V
H - 200, H-DPhe - Pro - Arg - Gly - Pro - Arg - Y~l - NHEt (L) Ihe synt~esis of this compound was carried out ~ccording to Scheme III.
(1) H-Y~l-NHEt.HCl (3 mmol) in CH2C12 at O , was acylated with 80c-Arg(Z2)-OPfp (2 mmol) in the pre3ence of Pr2N~t (2 m~ol) to yield~ nfter n ~tandard workup, the protected dipeptide ethylamide 25 (98%).
(2)(3) 25 (1.87 a ol) was dsprotected by treatment ~ith ~Cln20Ao and ncylated with 80c-Pro-OPfp (3 ~mol) in DMF at 0 in the presenco o~ ~Pr2NEt ~1.ô7 mmol). Standard ~orkup procedure ~nve the protected tripeptide ethylamide 27 l80%).
(4)(5) '27 (o.587 mmol) ~as deprotected ~ith HCl/EtOAc and reacted ln DMF ~olution at 0 in the pre~ence of Pr2NEt ~ith the Pfp ~tor 20 (prepared from 7 ~ith DCCI) to gi~e the protected pontnpeptide athyinmide 30 (95X).
(6)(7) ~0 (0.17 mmol) wns de-formylnted ~ith lN ~Clf~kne ~nd ~he hydrochloride ~alt ~ ~as isolated by e~aporation ~nd drying o~er ~OH pellets. It was then reacted in D~ at 0 ~ith the Pfp ester 11 $n the presence of Pr2NEt ~2 equi~nlents). A
mixture of epimers 32a and 32b was isolated from the reaction ~y stand~rd ~roced~res.

~322~78 (8)(9) ~pi~ers 32n ~nd 32b were ~epnrated by chromatogrnphy on ilica in MeOH-EtOAc (1:40) nnd the L-epimer 32a ~as hydrogenated in MeOH-AcOH-H20 (5~ over 5% Pd/C to give the Boc-protected hoptapeptide othyl~ide 33n.
(10) 33~ wn~ tr~nted with 2N HCl nt 22 ~or 2 hr~. Lyophili~ation yielded pure H-200.Tlc on silica: ~ 0.10 ln CHC13-MeOH-AcOH (6:2:1). An~lysis: Arg 1.10; Phe 0.99; Pro 2.07;
~al o.83.
Exnm~le YI
(L) R
H - 172, Boc - DPhe - Pro - Arg - Gly - Pro - 0~
~hi~ compound wa~ prepared accordinz to Scheme IV.
(1) Synthesis Or Boc-Arg(Z2)-H 18 was carried out accordin$ to the method of A.Ito et al., (Chem.Pharm.Bull.1~75~23.3001.) by reduction of ~oc-Arg~z2j-oMe with di-isobutyl-nluminium hydride (lBu2AlH) in toluene. The pure aldehyde 18 wa~ obtained 1rter puriflcnt~on by rnpid chromatography OQ ilica in 50%
yield.
(2) Preparation of H-Gly-Pro-OBzl, 19. Boc-6ly-0~ vas coupled via the mixed nnhydride( prepnred with i60but~1 chloroformate and N~J), to proline benzyl ester. The resulting Boc-Gly-Pro-OBzl wa5 trented with ~Cl/EtOAc to remove the Boc group nnd the HCl ~nlt of 19 thus formed ~as u~ed in the reductive ~lkylation step (3).
(3) Boc-Arg(Z2)RGly-Pro-OBzl, 20. The aldehy~e 18 (3 mmol) and ~-61y-Pro-OBzl 19 (3 mmol, obtained ~ro- the HCl snlt w1th NMM) in dry THF, ~ere allo~ed to re-ct in th~ prc once of 5A
~olec~lar Jieve (lOg) at -10 for 4 brs. ~he Schirr's base thus rormed wa3 reducod with Na GNBH3 (3 1 ole~) in ~ethanol at -1~. Pure product 20 w~s isolnted in 44~ ~-ield by chromntosraphy in EtOAc on Jilica.

~322~7~

(4) Boc-~rg(Z2)R ~ ly-Pro-OBzl, 21. The reducsd peptide 20 Troc (o.6 ~mol) in TH~ (40 ml) wns acylated ~ith 2,2,2-trichloro-- etho~ycArbonyl chloride (Troc-Cl, 0.7 ~mol) in the presence of NMM (0.7 mmol) at -15 . Silicn ~el chrom~togr~phy Or the crude product ~n EtoAc gave pure 21 in 58% yiold.

(5) 80c-DPhe-Pro-Ar8(Z2 ~ ly-Pro-OBml, 23.
Troc The protected rednced tripeptide 2t wns treated with HCl/
EtOAc to r~move the 80c protec*ing ~roup and the re ulting N-deprotected compound 22 (0.32 ;ol) w~ acylnted with 0 Boc-DPhe-Pro-OPfp (0.32 mmol) in dry nMF in the presence of Pr2N~t (0.32 ~mol). Silica chromatography of the crude product in EtOAc-benzene, produced pure 23 (45~).

(6) Removal of the Troc group from 23 to give intermediate 24.
.The fully protected reduced pentapeptide 23 (0.03 mmol) was dissolved in ~lacial acetic acid (o.8 ~1~ ~nd treated with zinc po~der (o.6 ~moL~innnatmo~phere Or nitrogen for 3 hr6.
Chro~atogrnphy of the crude product on silica gnve pure 24 in 47% yield.

(7) Prepar~tion of H-172. 2~ obtained in step (6) was dissolved in a mixture of HeOH-AcOH-H20 (5:1:t) and hydrogenated over 5~ Pd~C for 3 hrs. Hplc of the crude product on Partisil 10 ODS II in 64% HeOH-B20 containinS 0.2% ~ormic ucid gave pure H-172. Tlc in EtOAc-Py-AcOH-H20 130:20:6:1l) RF=0.25 on eilica. After hydroly~is t llO ~lB hrs ~ith 6N HCl:
Eound Pbe o.96, Pro 2.04.
, * Trade Mark .

~322~

Example VII
H-DPhe-Pro-Apa - Gly~Pro-NHEt 2-(41-kmidinophenyl)-alanine (Apa) was prep~red by an edaptation of the method of G. Wagner et al (Pharmazie, 1981.36.597) and was con~erted into the ~N-benzyloxy-carbonyl- ~N-formyl derivative by standard methods. The latter was subjected to the-sequence of reactions describet for the corresponding arginine derivative in ~
Schemes I and II to yield compound 40. Alternatively the reaction sequence may be carried out with 3- or 4-cyanophenylalanine and the side chai~ of the latter converted into the side chain of Apa or Amp at the end of the synthesis by the method of Wagner~
Example VIII
41 pNas-pro-Arg K Gly-Pro-~HEt Naphthalene-2-sulphoDql ~hloride (~Nas-Gl) was reacted with the sodium salt of L-proline under Schotten-Baumann conditions to yield ~Nas-Pro-OH. The latter was converted into the pentafluorophenyl ester and reacted with ~he partially protected tripeptide analogue 16.
(Scheme II) to give compound 41.
E~mple lt r~
42 H-Ipa-Pro-Ar~ Gly-~
t-~utyloxycarbonyl-4 L-io~ophenylalanine(Boc-Ipa-OH) was coupled to L-proline sodium salt via the pentafluoro-phenyl active ester, and the resulting Boc-Ipa-Pro-OH was then coupled to H-Arg(Z2) - Gly-piperidide. The L-epimer of the product (at the Arg '~-carbon) was separated by 1322~78 chromatography and deprotected, first by hydrogenolysis in the presence of palladium on charcoal, and then by treatment with 2M HCl to yield compound 42.

Example X

43 PhOCH2CO-Pro-Arg K Gly-Pro-NHEt L-Proline was ~-acylated with phenoxyacetyl chloride and the acyl compound coupled, via its pentafluorophenyl ester, to compound 16 (Scheme II) as described in Example VIII.

Example XI

44 H-DPhg-Pro-Arg -Gly-Pro-NHEt This compound was prepared according to the method described in Scheme II for Example IV, except that t-butyloxycarbonyl-D-phen~lglycine pentafluorophenyl ester (Boc-DPhg-OPfp) was used in place of Boc-DPhe-OPfp.

Example XII
Ac-NHC~ CH2-N(COPh)-CH( Pr)-CO-Arg - Gly-Pro-NHEt This analogue was synthesised according to the method described in SchRme VI.

1322~78 Example XIII

46 H2N-CH2CH2-N(~Nas)-CH( Pr)-CO-Arg - Gly-N ~
OOH
The above compound was synthesised according to Scheme VII.

Example XIV

47 DPhe-Pro-~rg K Gly-Pro-NH-CH2-CO

The synthesis of this cyclic peptide analogue is shown in Scheme VIII.

Example XV

K
48 C~Nal-Val-Arg - Gly-Pro-NH-CH2CH2-CO

The preparation of this cyclic peptide analogue is described in Scheme IX.

Example XVI

H 248 H-DPbe-Pro-Arg H Gly-Pro-NHEt (L~

The above analogue, which is believed to ~e in the S configuration at the carbon atom bearing the hydroxyl .

1322~78 group is prepared from the compound H 17g of Example IV
by reduction of the keto group with sodium cyanoboro-hydride and separation of the resulting mixture of R and S epimers.

SYNTHESIS SCHEMES
.

The synthesis schemes referred to above now follow.

1322~78 -- 24 ~
SCHEME.I (E~CamP1eS 1, 2, 7) R - _~CH2)3_N(Z)_CI=NH
NH-Z
H

R DP~CI N>~O
HCO-N~-CH-CO2H( 2 ) }~

.. CH2 - COC 1 .( 3 ) 1H2-C2CH2CC13, Et3N
(see below~

O DM~lPfPy N~OCOCH2CH2CO~CH2CC13 ,~0 ~ (4) ,~0( a H
(5)~py.AcoH~ ~ 4 (6) R Zn/T~- R
HCO-13H-CH-COCH2-CH2CO2CH2CC13 N~2 04 HCO-~H-CH-COCH2-CH2C02ll 6 DL 27% fl om 1 1007~ HCO-J~rg(Z2)~GlY~OH
D,L

Synthe si s of ,3 f~o ~ Cl CCH OH ~ ~ 1 2 C02H (COC1)2 3CH2-C02C}12CC13 --~ 3 75X 92~.

1322~78 Schem~ I (cont ' d) Boc-DPhe-OPfp H-Pro-O ~'a~ HCO-L ~ ly-O~

,¦,i, PfpOH/DCCI

¦ ( ~ ) H-~ro-OMe Boc-DPhe-Pro-OH HCO-Ar ~ ly-Pro-OMe 10 ¦Pfp-OH/DCCI 8 ¦ ~M HCl/MeOH

l l2 Boc-DPhe-Pro-OPfp H-~rg-Gly-Pro-OMe 30c-DPhe-Pro-Ar ~ ly-Pro-OMe 12~ ~ 12b - , ( 10) !OH

Boc-DPhe-Pro-Ar ~ ly-Pro-OH
13a ~ 13b Z/Pd ,', X ~
Boc-DPhe-Pro-Arg-Gly-Pro-OH ' D L

1322~8 ~ 26 ~
SCHEME II (Examples 3, 4, 7, 8, 10, 11) Boc-DPhe-OPfp H-Pro-O Nn HCO-Arg~-Gly-OH
V . 7 D,L ¦~) PfpOH/DCCI
-Pro-NHEt ~ Z2 Loc-DPhe-Pro-OH HCO-~rg-Gly-Pro-NHEt 10 ¦ Pfp-OH~D~CI (2) ¦ IMHCl~MeOH

Boc-DPhe-Pro-OPfp ~ ~-ArgX-Gly-Pro-NHEt Boc-DPhe-Pro-ArgK-Gly-Pro-NHEt 17~ 1 17b (D,L) (i) separation Or epimers ~ /~) (ii )H2/Pd Boc-DPhe-Pro-~rg-Gly-Pro-NHEt Boo-DPhe-Pro-Arg-Gly-Pro-NHEt H-170 tD) H-171 ~L) .
. . ZM ~Cl/h20 (5) 2M HClJH20 !
H-DPhe-Pro-Arg-Gly-Pro-NHEt . B-DPhe-Pro-~rg-Gly-Pro-NHEt ~ L) 132207~ .
- 27- .

SCHE~IE III .~Example 5) Boc-Arg-OPfp ~-Val NHEt-(1) . , .

Boc-Arg-Yal-NHEt 98%
(2, lZ
Boc-Pro-OPfp H-Arg-val-NHEt (3 . .

Boc-Pro-Arg-Yal-NHEt 27 80%
. H~ ..
(4) ~CO Ars~Gl OPf ~-Pr-Arg-Yal-NHEt 28 ~ 29 ~5~1 HCO-Ar~-Gly-~ro-Arg-Yal-NHEt 3 ~(D L) 95X
..

1322~178 ~ 28 --SCHE~ I~I (cont'd) ~6,1 lMHCl/MeOH

Boc-DPhe-Pro-OPfp H2-Arg-Gly-Pro-Arg-Yal-NHEt 31 (D L) Pr2NEt 22 ~ Z2 Boc-DPhe-Pro-Arg-Gly-Pro-Arg-Val-NHEt 32a 1 32b (L) (D) (8)l Separation of ~epi~ers on silica ~ .
32a(L) 32b(D) C

.. , ~ ' .

~oc-DPhe-Pro-~rg-Gly-Pro-Arg-Val-NHEt 33a IL) ( 10) ~HCl/H20 H2-DPhe-Pro-Arg-Gly-Pro-~rg-Val-NHEt Cl H-200 L epi~er ~322~7~

SC}EME IV (E~ample 6) sOc -Arg-OH
(L) 1 ' Boc-~rg-OH Boc-Gly-OH H-Pro-OBzl - IC~2N2 !~;~

Boc-Arg-OMe Boc-Gly-Pro-OBzl (1) i 1 8U2A1H ( 2 ) H
~ 2 Boc-Arg-H H-Gly-Pro-OBzl ~8 50% \ / 19 ,' \/

(i) ~ol. sieve (3) (ii) NaCNBH3 Boc-Arg---Gly-Pro-OBzl .. (20) 4~%
~ ~4~1qroc-Cl~NMM

Boc-Ar ~ y-Pro-OBzl 21 ~rOc 5~%

H-Ar ~ ly-Pro-OBzl 22 (L)$roc - 1322~78 St:HEME IV (cont'd) Boc-DPhe-Pro-OPfp ~-~rB r Gly-Pro-OBzl --- \ / 22 Troc ~5~¦ iPr2NEt ¦ DNF

Boc-DPhe-Pro-Ar ~ lY-Pro-OBzl 45% from 21 23 Sroc \ H2N-Et /AcOH ~ l2 Boc-DPhe-Pro-Ar ~ ly-Pro-~HEt l2 . Iroc Boc-DPhe-Pro-Ar ~ ly-Pro-OBzl Cd/AcOH

24 ~7~ ~ Z2 (7) H2/Pd Boc-DPhe-Pro-Ar ~ ly-Pro-~HEt .
Boc-DPhe-Pro-Ar~ R Gly-Pro-OH H2/Pd H 172 (L) 40%
-H~ ~oc-DPhe-Pro-Arg ~ ly-Pro-~HEt ~-DPhe-Pro-Ar ~ l~-Pro-O~ H

H-DPhe-Pro-Arg---Sly-Pro-~Et ~22~78 -SCHEME V (ExEmple 3~
12 H2C(COO~u )2, Z2 C2BU
Troc-Arg-CH2~r N~ , Troc-Arg-CH2-CH t 34 (L) 35 C2BU

H~
Pfp-OH z Troc- ~ gXGly OPf DCCI Troc-Ar~ ,~ly-OH
37 ~6 HproNHEt Troc-ArgKGly-ProNHEt A _H ~ H-Arg~Gly-ProNHEt /
/
~ Boc-DPhe-Pro-OPfp Eoc-DPhe-Pro-Arg~Gly-Pro-NHEt (L) ~322078 Scheme VI (Ex~nple 12) S9~1the8i8 of co~pound 45 ~c~I~2-C}IO }E-V~l ~ce \/
1) ~2 ' 2) NaC~3 ~c~i2CH2-1~-COOTce 1) PhCOCl 2) Zn/~cOa Z2 1~
~c~2ClI2~-CH-COOH H-~rg--Gly-Pro~EEt COF~ ~
DCCI/~B~

Zl 2 ~c~EI2-CH2~-C~[-CO--~g--G~-Pro-~
COPh . , H2/Pd-C
^. ~ .
~c~zC}12~1-CH-CO--~rg ~ Gl~r-Pro~Et CO~

Scheme VII (Example 13 Synthes~s of con~pou~d 46 Z~H-C~2~0 H-V~l ~ce \ /
1 ) -~I20 2) 17aC~3 Z~2CH2~ C}I_COO~ce 1 ) ~as-Cl 2) ~/~cO~l 1 ~ .
~ Z
Z~iH~C~E-CO~ H-~rg ~ Gl~
COOBzl DCCI/1~0:Bt ~' Z
~[2 2~Nl CH~Lg--Gl~
~ilas COOBzl H2/Pd-C

N~2C~I2-31JbH~ rg--Gly~¦
~,-Nas COOH

1~22978 -34~
Scheme VIII (Ex~ ~ le 14) Synthesi~ of compound 47 Roc-~rg ~ Gly-O~ h--Pro-Gly-O~ce ¦ Pfp~O~DCCI

~oc-~rg ~ Gly-Pro-Gly-O~ce ¦ HCl/diosan ~oc-DPhe-Pro-OPfp ~_Arg ~ Gly-Pro-Gly-OTce Et3~

Roc-DPhR-Pro~rg--Gly-~x~ y-Orce 1 ) Z~ cO~I
2) Pfp-O ~ CCI

~oc-DPhe-Pro-~rg ~ Gly-Prc~Gly-OPfp 1) CF~
2) E* ~, bi4h ~ilution 3) ~ -C

47 Doehe-Pro-~rg ~ G}y-Pro-Gly i322~7~

Scheme IX (Example 15) Synthesi~ Or compound 9~

~oc-~rg ~ Gly-O~ ~-Pro-~la-OTce \/
Z2 Pfp-O~VICCI

Boc-~rg ~ Gly-Pro-~la-OTce l~l/dio~an ~bc-Val-OPfp ~-~rg - Gly-Pro-~Ala-O~ce Et ¦ 2) ECl-dio~an ~oci~al-OPfp ~-V~ rg ~ Gly~Pr-pela-OTce \ ~ Et~
Z
~oc-Nal-Val-~rg ~ Gly-Pro-p~larO~ce 1 ) Z~
2~ Pf ~ I 4) Et ~, high dilution 3) qF~ 5) ~ d-C

g~ ~al-Val-~g ~ Gly-æ2D-p~la ~ . ..... .. . ... . . .. . . _ . ...

1322~78 BIOLOGICAL ACTIVITY
Compound~ were tested i~ vitro for the following nctivitie~, u~ing ~tandard procedure~:
.
(n) Inhibition of humnn thrombin hydrolysing the chromogenic ~ubstrnte 5-2238(for detail~ of the method, sec M.F.Scully ~nd V.V.~akkar, Clin.Chim.~cta 1977.~.595~. Series Or measurement~
were c~rried out u-~ing a number of different inhibitor concentrntions nnd at lea~t two different ~ubstrate concentrations. The inhibitory constant ~i wa5 determined 0 $raphicnlly, using a Dixon plot (M.Dixon, Biochem.J.1953.55.170) Ib) Prolongation of kaolin-cephalin clotting ti~e (KCCT; for method ~ee D.E.GAusten nnd I.L.Rhymes: ~Laboratory ~anual of Blood Coagulation", 81ackwell, Oxford 1975). Results have been expressed as the molar concentration of inhibitor required to double the KCCT.
(c) Inhibition of thrombin-induced platelet aggregation (for method ~ee G.Y.R.Born~ Nature 1962.19~.927).

Repre~entative results ~or E~amples I - VI are shown in the Tnble.

TABLE
Code ¦Example Ki for human ¦Conc.to double Ki for thrombin induced No ! No thrombin(~M) ¦ ~CC~ ~ M) Dlatelet nggregation ~ M) H-163i I 120 - ¦ 226 H-170III ! ' 0.3 25 H-172 VI 112 , 813 H-179IV 6 1 15 0.4 'H-200 V 3 !

-37_ i322078 Some of the compounds described in this specification have been tested in vivo for their ability to prolong clotting time and have shown marked activity. These tests were carried out in rabbits, using between 0.1-4 mg/Kg of the inhibitor and comparing the KCCT or the thrombin clotting time in blood samples taken before and at various time intervals after the administration of inhibitors.
H 179 for example prolongs thrombin clotting time from 25 to 220 seconds at 2.35 mg/Kg and from 25 to 280 seconds at 4.7 mg/Kg, and is comparable to heparin both in potency and in its in vivo half life in plasma (18 minutes;
heparin 15 minutes3.

- 38 - 132~7~

AbbreYiations used ~cOH Acetic ~cid Boc t-ButyloxycArbonyl iBu2AlH Di-isobutyl aluminium hydride Bzl Benzyl DCCI N,N'-Dicyclohexyl carbodiimide DCU NjN'-Dicyclohexylurea DMAP 4-Dimethylamino-pyridine DM~` Dimethylformamide DPECI N-dimethylaminopropyl-N'-ethyl-carbodi-- imide Et3N Triethylamine EtOAc Ethyl acetate hplc high p.erformance liquid chromatography K Keto isostere ~COCH2-M.A. Mixed anhydride MeOH Methanol NMM N-methylmorpholine nmr nuclear magnetic resonance Petrol Petroleum ether 60 - 80 Pfp Pentafluorophenyl iPr2NEt Di-isopropyl-ethylamine Py Pyridine R Reduced isostere -CH2-NH-tlc thin layer chromatography Troc 2,2,2-Trichloroethoxycarbonyl zl Benzyloxycarbonyl ( simply as Z in the examples) .

(Abbreviationæ - continued~

Amp 3-(3'-Amidinophenyl)-alanine Apa 3-t4~-Amidinophenyl)-alanine Ar Aryl: aromatic group, mono- or bicyclic Cha 3-Cyclohexyl-alanine HOBt l-Hydro~y-benzotriazole ~Nal 3-(1 -Naphthyl)-alanine pNal 3-(2'-~aphthyl)-alanine Phg 2-phenylglycine TFA Trifluoroacetic acid Tce 2,2,2-Trichloroethyl Tos Tosyl ~HF Tetrahydrofuran hplc High pressure liquid chromatography ~Nas Napht~alene-2-sulphonyl Ipa 4-Iodophe~ylalanine

Claims (8)

1. Polypeptide analogues of the formula (I) where X represents the following:
(i) H or an N-protecting group; or (ii) F, Cl, Br, I, -CF3, -OH, -OR or -R (R = C1 - C6 alkyl); or (iii) R-O-CO where R = t-butyl, benzyl, 2,2,2-trichloroethyl; or (iv) R6-SO2-wherein R6 = Ph, .alpha.-naphthyl, .beta.-naphthyl; or (v) one or more amino acyl residues either as such or in protected form bearing a group X of any of the above;
Y = absent, or glycine, or D- or L-phenylalanine or a lipophilic amino acid residue selected from the group consisting of Phg, Cha, .alpha.-Nal, .beta.-Nal and p-iodophenylalanyl Z = L- or D-proline or a ring homologue selected from the group consisting of azetidine-2-carboxylic acid and piperidine-2-carboxylic acid or L- or D-valine or N-methyl-alanine or glycine or Y and Z are as follows:

where the peptide bond -CO-NH- between Y and Z has been reduced (and protected) to give -CH2-N(X)-, X being a protective group as defined above 40a (1) (II) (2) (III) (3) (IV) where (i) or (ii) R2 = H, lower alkyl (C1-C4) or 1-hydroxyethyl, (iii) R3 = group X as defined above (iv) configuration at the asymmetric centres * is either R
or S
Arg or Pro and Arg may be absent, Pro may be hydroxy-proline and Arg may be substituted with Z1, Tos or -NO2 B = D or L-valine or D or L-proline or -NH-(CH2)n-CO-, where n=0-5, or absent W = -OH,-OR4 where R4 = lower alkyl (C1-C5) or an O-protecting group, -NH2-, -NHR5 or -N(R5)2 where R5 = lower alkyl (C1-C5), cycloalkyl (C3-C7)or cycloalkyl substituted with a group selected from carboxyl and alkyl (C1-C5) group(s) or an N-protecting group, or a group with one or more further amino acyl residues, or B-W represents an aminoalcohol derivative of B as such or in protected form and where further in said compounds of formula I
the two terminals are optionally linked by a peptide bond to form a cyclic structure.

2. Polypeptide analogues according to claim 1, wherein X = H, lower alkyl (C1-C5), lower acyl (C1-C5), R-O-CO-where R = t-butyl, benzyl, 2,2,2-trichloroethyl, or an amino acyl residue either as such or in a protected form Y = D- or L-phenylalanine, or glycine Z = L- or D-proline, or L- or D-valine In A:

or or or or or with n = 2, 3 or 4 R2 = H, methyl, isopropyl, sec-butyl, iso-butyl or 1-hydroxy-ethyl R3 = H, lower aliphatic acyl C1-C5 or lower alkyl C1-C5 configuration at the asymmetric centres * is either R or S
B = D- or L-valine, or D- or L-proline, or absent W = -OH, or -OR4 where R4 a lower alkyl C1-C5, or -NH2, or -NHR5 or N(R5)2 where R5 = lower alkyl (C1-C5) or (R5)2 = lower cycloalkyl -(CH2)n- with n = 3, 4 or 5.

3. Polypeptide analogues according to claim 1 wherein A is the 'keto' isostere (II).

4. Polypeptide analogues according to claim 1, modified by isosteric replacement of one or more remaining peptide bonds by 'keto', 'hydroxy' or 'reduced' isosteric links.

5. Polypeptide analogues according to claim 1, in protected form at one or more remaining amino, imino, amide (including peptide), guanidino, hydroxyl, carboxyl or other functional groups.

6. The following polypeptide analogues or or or or or or

7. The following polypeptide analogues or or or or

8. Polypeptide analogues according to claim 1, wherein:
X represent6 the following:
(i) H or an N-protecting qroup; or (ii) F, Cl, Br, I, -CF3, -OH, -OR or -R (R = C1 - C6 alkyl); or (iii) R-O-CO where R = t-butyl, benzyl, 2,2,2-trichloroethyl; or (iv) R6-SO2-wherein R6 = Ph, .alpha.-naphthyl, .beta.-naphthyl; or (v) one or more amino acyl residues either as such or in protected form bearing a group X of any of the above;
Y = D- or L-phenylalanine, or glycine, Z = L- or D-proline, or L- or D-valine In A:

with n = 2, 3 or 4 R2 = H, methyl, isopropyl, sec-butyl, iso-butyl or 1-hydroxy-ethyl R3 = H, lower aliphatic acyl C1-C5 or lower alkyl C1-C5, or t-butyl, benzyl, or 2,2,2-trichloroethyl configuration at the asymmetric centres * is either R or S
B = D- or L-valine, or D- or L-proline, or absent W = -OH, or -OR4 where R4 = lower alkyl C1-C5, or -NH2, or -NHR5 or N(R5)2 where R5 = lower alkyl (C1-C5) or alternatively (R5)2 = lower cycloalkyl -(CH2)n- with n = 3, 4 or 5.